This invention pertains to the field of medicine and medical biology. The specific sub-area of medicine and medical biology is the art of the in vitro culture of mammalian tissue cells. Many nutrient culture mediums have been developed and are, to a large extent, specifically designed to permit prolonged and sustained growth of specific types of cells, such as those derived from skin, from tumors, and from various other tissues in mammalian species. These are grown by primary culture systems as explants, monolayers or suspension cultures or secondary cell lines. However, there is no culture system or nutrient media available at the present which will permit prolonged growth and proliferation of human anterior pituitary cells in sufficient number to provide a source of large amounts of growth hormone and other pituitary hormones. This lack of success is related to many factors, among which are (1) limited knowledge concerning the specific nutrients for the specific cells, (2) the general trend for the pituitary hormone-producing cells to lose their function of producing hormones, (3) death of cells after a few days to a few weeks, and (4) transformation of cells to non-functioning fibroblastic or fibroblastoid type cells.
Some of the previous studies which indicate the difficulties encountered are as follows: Animal and human pituitary cells have been maintained as explants in cultures and are known to release their hormones for variable periods of time. Continuous cell lines derived from induced tumors in rats may proliferate for several weeks or a few months and are able to secrete some amounts of rat growth hormone. (Takemoto, H., Yokoro, K., Furth, J. and Cohen, A. I., Cancer Res., 22:917-924, 1962; Tashjian, A. H. and Hoyt, R. F., Jr., In Molecular Genetics and Developmental Biology, edited by M. Sussman, pp. 353-387, Prentice-Hall, New Jersey, 1972). Tumors of the human pituitary gland have also been maintained in culture and may secrete human growth hormone into the culture media for variable periods of time (Kohler, P. O., Bridson, W. E., Rayford, P. L. and Kahler, S. E., Metabolism, 18:782, 1969; Batzdorf, U., Gold, V., Matthews, N. and Brown, J., Neurosurgery 34:741, 1971; Peillon, F., Gourmelen, M., Donnadieu, M., Brandi, A., Sevaux, D. and Pham Huu Trung, M. T., Acta Endocrinol., 79:217-229, 1975) whereas normal human pituitary cells in culture survive and produce hormone only for a relatively brief period of time. The number of cells and the amount of hormone produced in such systems decline rapidly with time (Vidal-Tixier, A., Gourdji, D. and Tougard, C., Intern. Rev. Cytol., 41:173-239, 1975). Human fetal pituitary cells secreting growth hormone have also been cultured for several days but ultimately die or are transformed into fibroblast type cells (Gailani, S. D., Nussbaum, A., McDougall, W. J. and McLimans, W. F., Proc. Soc. Exp. Biol. and Med., 134:27-32, 1970).
The above described previously reported results by others were obtained by a culture procedure in which either small explanted pieces of tissue or a monolayer of cells were grown in small volumes in medium (Peillon et al, Vidal-Tixier et al and Gailani et al, cited above), into which only a small amount of growth hormone was released or secreted by the cells. The nutrient mediums used in these cultures have been unable to permit replication of the growth hormone producing cells. In all of these previously reported studies, the rate of cell multiplication and the amount of hormone produced, while persisting for a brief period of time, rather rapidly declines (Peillon et al). The culture systems or media used did not adequately meet the growth and metabolic needs of the cells as conversion to fibroblastoid cells, which are non-hormone producing or a loss of the growth hormone synthesis capacity in the cells or death of the cells occurred.
The literature concerning the art of tissue culture procedure and of nutrient medium developed is very voluminous and particularly extensive over the last 20 to 25 years. The hormones produced by the anterior pituitary in man include human growth hormone, ACTH (adrenocorticotrophic hormone), LH (lutenizing hormone), FSH (follicle stimulating hormone), PRL or LTH (prolactin or luteotrophic hormone) and TSH (thyrotrophic hormone). The inventors have been unable to find evidence of a previously described culture system or nutrient medium which permits the growth and proliferation of human pituitary hormone producing cells in sufficient amounts and over a long period of time to produce adequate amounts of hormone which can be biochemically extracted and used to treat hormone deficient states in man.